Team:Cambridge-JIC/Chromoproteins
From 2014.igem.org
(Difference between revisions)
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<h3>E-Coli transformation and Plasmid Purification</h3> | <h3>E-Coli transformation and Plasmid Purification</h3> | ||
- | Attempt 1: | + | <h4>Attempt 1: </h4> |
Transformation Date: 21.07.2014 | Transformation Date: 21.07.2014 | ||
Comments: Left to grow on Kan+ LB agar plates O/N at 37 C, with a negative (Gibson) control. | Comments: Left to grow on Kan+ LB agar plates O/N at 37 C, with a negative (Gibson) control. | ||
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The concentrations of the plasmid DNAs were read using Nanodrop and documented in the DNA library (A10, A11, A12). | The concentrations of the plasmid DNAs were read using Nanodrop and documented in the DNA library (A10, A11, A12). | ||
- | Attempt 2: | + | <h4>Attempt 2: </h4> |
Transformation Date: 22.07.2014 | Transformation Date: 22.07.2014 | ||
Comments: Left to grow on Kan+ LB agar plates O/N at 37 C. A positive control (p35sRLti) was also used. | Comments: Left to grow on Kan+ LB agar plates O/N at 37 C. A positive control (p35sRLti) was also used. | ||
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After colony PCR, selected colonies were picked from each, grown O/N and the plasmids were then extracted by miniprep (DNA Library A13, A14, A15, A16). | After colony PCR, selected colonies were picked from each, grown O/N and the plasmids were then extracted by miniprep (DNA Library A13, A14, A15, A16). | ||
- | Attempt 3: | + | <h4>Attempt 3: </h4> |
Transformation Date: 25.07.2014 | Transformation Date: 25.07.2014 | ||
Comments: Left to grow on Kan+ LB agar plates O/N at 37 C. A positive control (p35sRLti) was also used, in addition to the Gibson negative control. The plates were removed from | Comments: Left to grow on Kan+ LB agar plates O/N at 37 C. A positive control (p35sRLti) was also used, in addition to the Gibson negative control. The plates were removed from | ||
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<h4>Colony PCR</h4> | <h4>Colony PCR</h4> | ||
- | Attempt 1: | + | <h4>Attempt 1: </h4> |
Date:24.07.2014 | Date:24.07.2014 | ||
Comments: The eforRed (cyto) colonies showed a band of approx. 1kb, agreeing with the predicted 1042bp. asPink colony bands resembled the bands of the p35sRLti control. However, the band was close enough to the predicted 1303bp for us to pick one of the colonies for O/N culture. | Comments: The eforRed (cyto) colonies showed a band of approx. 1kb, agreeing with the predicted 1042bp. asPink colony bands resembled the bands of the p35sRLti control. However, the band was close enough to the predicted 1303bp for us to pick one of the colonies for O/N culture. | ||
[PHOTO 1 SCAN HERE] | [PHOTO 1 SCAN HERE] | ||
- | Attempt 2: | + | <h4>Attempt 2: </h4> |
Date:25.07.2014 | Date:25.07.2014 | ||
Comments: The predicted band size for tsPurple (cyto) is 1045bp, eforRed (cyto) 1042bp and amilCP (nucl) 1270bp. Colonies which fit this prediction most closely were chosen for further analysis. | Comments: The predicted band size for tsPurple (cyto) is 1045bp, eforRed (cyto) 1042bp and amilCP (nucl) 1270bp. Colonies which fit this prediction most closely were chosen for further analysis. | ||
[PHOTO 2 SCAN HERE] | [PHOTO 2 SCAN HERE] | ||
- | Attempt 3: | + | <h4>Attempt 3: </h4> |
Date:28.07.2014 | Date:28.07.2014 | ||
Comments: Due to a large degree of smudging present on the gel, all colonies were chosen for future analysis (digest). | Comments: Due to a large degree of smudging present on the gel, all colonies were chosen for future analysis (digest). | ||
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<h3>Agrobacteria transformation</h3> | <h3>Agrobacteria transformation</h3> | ||
- | Attempt 1: | + | <h4>Attempt 1: </h4> |
Date: 29.07.2013 | Date: 29.07.2013 | ||
Comments: Electroporation was done on our stock of competent Agrobacteria (protocol from the Plants Dept - http://www.plantsci.cam.ac.uk/research/jillharrison/protocols/cloning/preparation-of-agrobacterium-electrocompetent.pdf/view), following the Abrobacteria Transformation protocol. All 13 constructs were transformed, | Comments: Electroporation was done on our stock of competent Agrobacteria (protocol from the Plants Dept - http://www.plantsci.cam.ac.uk/research/jillharrison/protocols/cloning/preparation-of-agrobacterium-electrocompetent.pdf/view), following the Abrobacteria Transformation protocol. All 13 constructs were transformed, | ||
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<h3>Spore preparation</h3> | <h3>Spore preparation</h3> | ||
- | Attempt 1: | + | <h4>Attempt 1: </h4> |
Start date/time: | Start date/time: | ||
End date/time: | End date/time: |
Revision as of 11:44, 12 August 2014
Gibson
Attempt 1:
- Date: 21.07.2014
- Comments: Attempted to assemble all 7 constructs. The Gibson assembly was done following the protocol.
Fragments 1 and 2 were used for all constructs since they represent the pGreen backbone. Nuclearly-localised constructs (N7) also required the addition of 3 (i.e. N7 fragment). The other fragments were added to each constuct reaction specifically:
- eforRed (cyto) - 4
- tsPurple (cyto) - 7
- eforRed (nucl) - 5
- amilCP (nucl) - 6
- tsPurple (nucl) - 8
- asPink (nucl) - 9
- aeBlue (nucl) - 10
Attempt 2:
- Date: 22.07.2014
- Comments: Attempted to assemble all 7 constructs. The Gibson assembly protocol was slightly altered, i.e. 1ul of each sample was added to the reaction tube instead of 0.5ul. To account for this change, the amount of master mix added was scaled to 3x the final volume.
- The rest is identical to Attempt 1.
Attempt 3:
- Date: 23.07.2014
- Comments: Attempted to assemble ONLY nuclear-localised constructs. The Gibson assembly was done following the protocol.The higher-concentration backbone fragments from the cpIII aplification series were used in conjunction with cpII chromoprotein fragments.